The rate of lactate production from glucose in hearts is not altered by per-deuteration of glucose

Abstract

This study was designed to determine whether perdeuterated glucose experiences a kinetic isotope effect (KIE) as glucose passes through glycolysis and is further oxidized in the tricarboxylic acid (TCA) cycle. Metabolism of deuterated glucose was investigated in two groups of perfused rat hearts. The control group was supplied with a 1:1 mixture of [U-13C6]glucose and [1,6-13C2]glucose, while the experimental group received [U-13C6,U-2H7]glucose and [1,6-13C2]glucose. Tissue extracts were analyzed by 1H, 2H and proton-decoupled 13C NMR spectroscopy. Extensive 2H-13C scalar coupling plus chemical shift isotope effects were observed in the proton-decoupled 13C NMR spectra of lactate, alanine and glutamate. A small but measureable (~8%) difference in the rate of conversion of [U-13C6]glucose vs. [1,6-13C2]glucose to lactate, likely reflecting rates of C–C bond breakage in the aldolase reaction, but conversion of [U-13C6]glucose versus [U-13C6,U-2H7]glucose to lactate did not differ. This shows that the presence of deuterium in glucose does not alter glycolytic flux. However, there were two distinct effects of deuteration on metabolism of glucose to alanine and oxidation of glucose in the TCA. First, alanine undergoes extensive exchange of methyl deuterons with solvent protons in the alanine amino transferase reaction. Second, there is a substantial kinetic isotope effect in metabolism of [U-13C6,U-2H7]glucose to alanine and glutamate. In the presence of [U-13C6,U-2H7]glucose, alanine and lactate are not in rapid exchange with the same pool of pyruvate. These studies indicate that the appearance of hyperpolarized 13C-lactate from hyperpolarized [U-13C6,U-2H7]glucose is not substantially influenced by a deuterium kinetic isotope effect.

In vivo 13C and 1H‐[13C] MRS studies of neuroenergetics and neurotransmitter cycling, applications to neurological and psychiatric disease and brain cancer

“…In addition, due to the greater isotope weight ratio of 2 H/ 1 H versus 13 C/ 12 C, isotope effects that could potentially slow down chemical reactions may result in underestimates of metabolic rates. Although this problem has been addressed in other systems, for example in whole body stable isotope turnover measurements using deuterated H 2 O, and in perfused rat heart , further work is needed to determine how it impacts quantifying brain metabolism.…”

Effects of deuteration on transamination and oxidation of hyperpolarized 13C-Pyruvate in the isolated heart

“…The distribution of species [A] through [F] (Fig. lb) were calculated using methods reported previously [9]. Here, the analysis is performed using the ratio of the quartet area (representing [U-13 C 3 ]pyruvate) versus the area of the individual singlet species [C] though [F] in the C2 resonance of lactate and alanine in the 13 C NMR spectra [9].…”

“…The 2 H nucleus is quadrupolar, so it increases the complexity of 13 C NMR spectra for carbon atoms with one or more directly attached deuterium atoms. The mass differences between 1 H and 2 H also induces a chemical shift in the 13 C NMR spectrum, typically, a onebond chemical shift of 0.25 ppm per 2 H, and a two-bond shift of about 0.05 ppm per 2 H is observed [9]. A third factor is exchange of 2 H labels in a metabolite with 1 H in tissue water in vivo.…”

“…These factors were previously examined during metabolism of [U-13 C 6 , U-2 H 7 ]glucose in isolated hearts. One surprising finding was the dramatic difference in deuterium distribution in alanine compared to lactate, which can only arise if pyruvate ↔ alanine exchange and pyruvate ↔ lactate exchange occur in distinctly different cellular compartments [9]. Although no kinetic isotope effects were observed in the conversion of glucose to lactate, a kinetic difference was observed in the conversion of pyruvate → alanine (catalyzed by ALT) and conversion of pyruvate → glutamate (via the TCA cycle).…”

Probing carbohydrate metabolism using hyperpolarized 13C‐labeled molecules

“…It is worth noting that, although the use of deuterated metabolic substrates is expected to induce deuterium isotope effects on the metabolic processes, no such effect was observed with deuterated glucose derivatives . This was attributed to the multistep nature of glycolysis as well as to the small contribution of the bond breaking steps to the kinetic control of the enzymatic steps …”